A commonly encountered situation in genetic analysis entails the need to identify a low percent of variant DNA sequences (‘target sequences’) in the presence of a large excess of non-variant sequences (‘reference sequences’). Examples for such situations include: (a) identification and sequencing of a few mutated alleles in the presence of a large excess of normal alleles; (b) identification of a few methylated alleles in the presence of a large excess of unmethylated alleles (or vice versa) in epigenetic analysis; (c) detection of low levels of heteroplasmy in mitochondrial DNA; (d) detection of drug-resistant quasi-species in viral infections and (e) identification of tumor-circulating DNA in blood of cancer patients (where people are suspected of having cancer, to track the success of cancer treatment or to detect relapse) in the presence of a large excess of wild-type alleles.
The inventor of the present application has previously described COLD-PCR methods for enriching the concentration of low abundance alleles in a sample PCR reaction mixture; see published patent PCT application entitled “Enrichment of a Target Sequence”, International Application No. PCT/US2008/009248, now U.S. Ser. No. 12/671,295, by Gerassimos Makrigiorgos and assigned to the assignee of the present invention. The described COLD-PCR enrichment methods are based on a modified nucleic acid amplification protocol which incubates the reaction mixture at a critical denaturing temperature “Tc”. The prior patent application discloses two formats of COLD-PCR, namely full COLD-PCR and fast COLD-PCR.
In full COLD-PCR, the reaction mixture is subjected to a first denaturation temperature (e.g., 94° C.) which is chosen well above the melting temperature for the reference (e.g., wild-type) and target (e.g., mutant) sequences similar to conventional PCR. Then, the mixture is cooled slowly to facilitate the formation of reference-target heteroduplexes by hybridization. Steady lowering of the temperature in a controlled manner from 94° C. to 70° C. over an 8 minute time period is typical to assure proper hybridization. Alternatively, the temperature is rapidly lowered to 70° C. and retained at this temperature for 8 min to assure proper hybridization. Once cooled, the reaction mixture contains not only reference-target heteroduplexes but also reference-reference homoduplexes (and to a lesser extent target-target homoduplexes). When the target sequence and reference sequence cross hybridize, minor sequence differences of one or more single nucleotide mismatches or insertions or deletions anywhere along a short (e.g., <200 bp) double stranded DNA sequence will generate a small but predictable change in the melting temperature (Tm) for that sequence (Lipsky, R. H., et al. (2001) Clin Chem, 47, 635-644; Liew, M., et al. (2004) Clin Chem, 50, 1156-1164). Depending on the exact sequence context and position of the mismatch, melting temperature changes of 0.1-20° C., are contemplated. Full COLD-PCR, as described in the above referred patent application, is premised on the difference in melting temperature between the double stranded reference sequence and the hybridized reference-target heteroduplexes. After cooling down to form reference-target heteroduplexes, the reaction mixture is incubated at a critical denaturing temperature (Tc), which is chosen to be less than the melting temperature for the double stranded reference sequence and higher than the lower melting temperature of the reference-target heteroduplexes, thereby preferentially denaturing the cross hybridized target-reference heteroduplexes over the reference-reference homoduplexes.
The critical denaturing temperature (Tc) is a temperature below which PCR efficiency drops abruptly for the reference nucleic acid sequence (yet sufficient to facilitate denaturation of the reference-target heteroduplexes). For example, a 167 bp p53 sequence amplifies well if the PCR denaturing temperature is set at 87° C., amplifies modestly at 86.5° C. and yields no detectable product if PCR denaturation is set at 86° C. or less. Therefore, in this example Tc˜86.5° C. After intermediate incubation at the critical denaturing temperature (Tc), the primers are annealed to the denatured target and reference strands from the denatured heteroduplexes and extended by a polymerase, thus enriching the concentration of the target sequence relative to the reference sequence. One of the advantages of full COLD-PCR is that the same primer pair is used for both target and reference sequences.
Fast COLD-PCR, as described in the above referred patent application, is premised on there being a difference in melting temperature between the double stranded reference sequence (e.g., wild-type sequence) and the double stranded target sequence mutant sequence). In particular, the melting temperature of the target sequence must be lower than the reference sequence. The critical denaturing temperature (Tc) in fast COLD-PCR is a temperature below which PCR efficiency drops abruptly for the double stranded reference nucleic acid sequence, yet is still sufficient to facilitate denaturation of the double stranded target sequence. During the fast COLD-PCR enrichment cycle, the reaction mixture is not subjected to denaturation at a temperature (e.g., 94° C.) above the melting temperature of the reference sequence as in the first step of the full COLD-PCR cycle. Rather, the reaction mixture is incubated at a critical denaturing temperature (e.g., Tc=83.5° C.), which is chosen either (a) to be less than the melting temperature for the double stranded reference sequence and higher than the lower melting temperature of the double stranded target sequence, or; (b) to be lower than the Tm of both reference and target sequences, whilst still creating a differential between the degree of denaturation of reference and target sequences. After incubation at the critical denaturing temperature (Tc), the primers are annealed to the denatured target strands and extended by a polymerase, thus enriching the concentration of the target sequence relative to the reference sequence. Again, the same primer pair is used for both target and reference sequences.
Enrichment via full COLD-PCR has been found to be relatively inefficient, and time consuming, compared to enrichment via fast COLD-PCR. However, the use of fast COLD-PCR is limited to applications in which the melting temperature of the double stranded target sequence is suitably less than the melting temperature for the double stranded reference sequence. For example, mutations will not be detectable in sequencing data for a sample with a low abundance of mutant sequences that has been subjected to fast COLD-PCR if the melting temperature of the mutant sequence is the same or higher than the melting temperature of the wild-type sequence. Therefore, it is desired to improve the efficacy and rate of the full COLD-PCR cycle.
It is believed that the relative inefficiency of full COLD-PCR is due primarily to the paucity of heteroduplexes formed particularly during the early cycles of full COLD-PCR. Even if slow cool down during the hybridization step is optimized (e.g., steadily cool down for 8 minutes from 94° C. to 70° C.), the very low concentration of target (e.g. mutant) strands especially during early cycles reduces the ability to form heteroduplexes. Increasing the time for hybridization cool down is not desired, and in any event has not been found to be particularly effective to improve enrichment. Another reason that full COLD-PCR may be relatively less efficient than fast COLD-PCR is that the amplicons during later cycles of full COLD-PCR have a propensity to reform their homoduplexes rather than form heteroduplexes.
One object of the present invention is to improve the efficiency of heteroduplex formation in the early cycles of full COLD-PCR. Another object is to decrease the overall cycle time for full COLD-PCR.